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Oncogenic and Rasopathy-Associated K-RAS Mutations Relieve Membrane-Dependent Occlusion of the Effector-Binding Site

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Oncogenic and Rasopathy-Associated K-RAS Mutations Relieve Membrane-Dependent Occlusion of the Effector-Binding Site Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site Mohammad T. Mazhab-Jafaria,1, Christopher B. Marshalla, Matthew J. Smitha, Geneviève M. C. Gasmi-Seabrooka, Peter B. Stathopulosa,2, Fuyuhiko Inagakib, Lewis E. Kayc,d, Benjamin G. Neela, and Mitsuhiko Ikuraa,3 aDepartment of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9; bFaculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan; cDepartments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and dProgram in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 Edited by Thomas C. Pochapsky, Brandeis University, Waltham, MA, and accepted by the Editorial Board April 6, 2015 (received for review October 17, 2014) K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a on the anionic membrane and that these orientations could be prenylated, membrane-associated GTPase protein that is a critical influenced by the bound nucleotide. Here we present high-resolu- switch for the propagation of growth factor signaling pathways to tion NMR-derived models of the dynamic interactions between diverse effector proteins, including rapidly accelerated fibrosar- K-RAS4B and the lipid bilayer and how they can be impacted by coma (RAF) kinases and RAS-related protein guanine nucleotide disease-associated mutations or interactions with effector proteins. dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clin- Results ical outcome, whereas germ-line mutations are associated with de- K-RAS4B Activation State Determines Population of Two Major velopmental syndromes. However, it is not known how these Orientations on Lipid Bilayer. Recent developments in solution mutations affect K-RAS association with biological membranes or NMR technology have made it possible to study integral membrane whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid proteins and those anchored to membranes (7). To overcome the BIOPHYSICS AND bilayer-anchored K-RAS4B and its interactions with effector protein inherent challenges associated with solution NMR studies of COMPUTATIONAL BIOLOGY 13 RAS-binding domains (RBDs). Unexpectedly, we found that the membrane-associated proteins, we used selective C-labeling of effector-binding region of activated K-RAS4B is occluded by inter- action with the membrane in one of the NMR-observable, and thus Significance highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B KRAS (Kirsten rat sarcoma viral oncogene homolog) is frequently from the occluded state to RBD-specific effector-bound states. Im- mutated in pancreatic, colon, and lung tumors, which predicts portantly, we found that two Noonan syndrome-associated muta- poor clinical outcome, whereas germ-line mutations are associ- tions, K5N and D153V, which do not affect the GTPase cycle, relieve ated with developmental disorders, including Noonan syndrome. the occluded orientation by directly altering the electrostatics of two Although K-RAS is an attractive anticancer target, no clinically membrane interaction surfaces. Similarly, the most frequent KRAS successful inhibitors are available. Most disease-associated mu- oncogenic mutation G12D also drives K-RAS4B toward an exposed tations elevate the activated GTP-bound form of KRAS; however, configuration. Further, the D153V and G12D mutations increase the some remain unexplained. KRAS signals from cellular mem- rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. branes; however, our studies revealed that its association with We revealed a mechanism of K-RAS4B autoinhibition by membrane the membrane surface sequesters its binding site for effector sequestration of its effector-binding site, which can be disrupted by proteins, hampering signaling. Some disease-associated KRAS disease-associated mutations. Stabilizing the autoinhibitory interac- mutations disrupt this autoinhibition, identifying a new gain- tions between K-RAS4B and the membrane could be an attractive of-function mechanism and explaining how certain Noonan syn- target for anticancer drug discovery. drome mutations activate K-RAS signaling. Importantly, these findings open new avenues for therapeutic strategies to target KRAS | nuclear magnetic resonance | lipid bilayer nanodisc | oncogenic oncogenic K-RAS through stabilizing autoinhibitory interactions mutation | Noonan syndrome with the membrane. he K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) Author contributions: M.T.M.-J., C.B.M., M.J.S., G.M.C.G.-S., P.B.S., F.I., L.E.K., B.G.N., and KRAS M.I. designed research; M.T.M.-J. performed research; M.J.S., F.I., and B.G.N. contributed Tprotein product of the gene undergoes posttranslational new reagents/analytic tools; M.T.M.-J., C.B.M., G.M.C.G.-S., P.B.S., L.E.K., and M.I. analyzed farnesylation and C-terminal processing, which, in conjunction with data; M.T.M.-J., C.B.M., and M.I. wrote the paper. a poly-basic hypervariable region (HVR), targets K-RAS4B to The authors declare no conflict of interest. anionic lipid rafts on the intracellular side of the plasma mem- This article is a PNAS Direct Submission. T.C.P. is a guest editor invited by the Editorial brane (Fig. 1A) (1). This localization is essential for K-RAS4B Board. function and enhances signaling fidelity (2). Although the signif- Data deposition: The atomic coordinates and structure factors have been deposited in the icance of membrane tethering of K-RAS4B is well appreciated, a Protein Data Bank, www.pdb.org (PDB ID codes: 2MSC, 2MSD, and 2MSE), and in the high-resolution map of how K-RAS4B interacts with the mem- Biological Magnetic Resonance Bank, www.bmrb.wisc.edu (BMRB entry ID codes: 25114, 25115, and 25116). brane is lacking. Because membrane-anchored RAS presents a 1Present address: Program in Molecular Structure and Function, Hospital for Sick Children, major challenge to crystallization, current structural insights into the Toronto, ON, Canada M5G 1X8. behavior of membrane-anchored RAS have come from a variety of 2Present address: Department of Physiology and Pharmacology, Schulich School of Med- lower-resolution techniques including in vivo FRET-based studies icine & Dentistry, London, ON, Canada N6A 5C1. (3), fluorescence and infrared spectroscopic studies (4–6), and in 3To whom correspondence should be addressed. Email: [email protected]. silico models (3). These pioneering studies suggested that the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. K-RAS4B GTPase domain adopts certain preferred orientations 1073/pnas.1419895112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1419895112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A All 11 isoleucines are located <4 Å from the protein surface and are well distributed throughout the protein to provide excellent probes for molecular interactions. To unambiguously identify re- gions of the GTPase domain interacting with or in close proximity to the lipid bilayer, we prepared nanodiscs incorporating lipids + conjugated to the paramagnetic ion gadolinium (Gd3 )and measured broadening of the K-RAS4B Ile Cδ resonances induced by paramagnetic relaxation enhancement (PRE) (10). Mapping the PRE-broadened Ile probes on the structure of K-RAS4B (Fig. 2 A and B and Table S1) revealed that not all of the observed PRE-derived constraints can be simultaneously satisfied by a sin- gle interface, indicative of equilibrium between at least two ori- entations with distinct sites of membrane association. A comparison of the PRE profiles of the active and inactive forms of K-RAS4B reveals that Ile36 and Ile139 are the residues most sensitive to the activation state (Fig. 2A). Ile36 is located at the C terminus of switch I in close proximity to switch II, and Ile139 is on the opposite side of the GTPase domain in proximity to helix-α4, suggesting that nucleotide loading shifts the equi- librium of membrane association of these two surfaces. To construct structural models of membrane-bound K-RAS4B and identify preferred orientations of the active and inactive forms, we carried out high ambiguity-driven biomolecular docking (HADDOCK) simulations (11) (Table S2) using distance re- B straints derived from the PRE experiments (10) (Fig. 2 A and B). The ensemble of models computed by HADDOCK for activated and inactive K-RAS4B each clearly exhibit two distinct clusters of orientations (Fig. 2C and Fig. S2B), consistent with orienta- tional flexibility of the K-RAS4B GTPase domain (6); however, the relative population of each cluster was dependent on the activation state (Table S2). In the cluster that predominates in the GDP-bound form, most of the K-RAS4B helices are oriented parallel to the membrane, and a surface comprised of the N terminus of β1 (N-β1), α4, β6, α5, and the loop connecting β2 and β3 interfaces with the membrane (Fig. 2 C and D). This surface, located opposite to switch I, is therefore designated the α-interface. By contrast, the helices are oriented semiperpendicular to the membrane in the major cluster for activated K-RAS4B- GMPPNP (a nonhydrolyzable analog of GTP), and the mem- brane interface is formed
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